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Dazhi Lai

Publications and source records attributed to Dazhi Lai.

3 recordsLinked to original sources
PubMed2004

Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

Introns are present in some human pre-tRNAs. They are spliced out during the maturation processes of pre-tRNAs in a way that is irrelevant to their specific nucleotide sequences. This unique characteristic of tRNA splicing can be used for generation of small antisense RNAs by replacing the intron sequences with corresponding antisense sequences. In this work, the intron sequence of human pre-tRNAtyr gene was replaced with a 20 bp antisense sequence targeted to the 5' coding region of cyclin D1, a molecule that was over-expressed in many malignant proliferating cells. Under the control of U6 SnRNA promoter to further enhance transcription efficiency of the modified pre-tRNAtyr gene and subsequent antisense generation, the antisense RNA exhibited obvious suppression of cyclin D1 expression in H22 hepatoma cells. The growth of H22-transplanted tumors in mice was significantly inhibited when treated with naked plasmid DNA harboring the cyclin D1 antisense RNA generating cassette. Such tumor growth inhibition might be due to apoptosis caused by reduced cyclin D1 expression as revealed by immunohistochemical analysis of tumor samples.

Alternative Splicing↗
PubMed2004

Blocking caspase-3 activity with a U6 SnRNA promoter-driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-beta.

Apoptosis responding to various insults in bioreactors was observed to reduce cell viability and prevent cells from growing to high density. Inhibition of apoptosis in different ways has proved to be effective in keeping cells viable in high density and result in higher recombinant protein production. In apoptosis, death signals activate a family of proteinases, namely caspases, in a cascade and ultimately activate the final effector proteinase, caspase-3, which cleaves various substrates and drives the cells irreversibly to death. Caspase-3 is the executioner of an apoptotic cell and thus plays a central role in apoptosis. Therefore inhibition of caspase-3 may provide an effective way for apoptosis prevention. In this study, we designed a ribozyme targeted at the 451 nt of hamster caspase-3's open reading frame (ORF) and the ribozyme was proved to be effective in cleaving caspase-3 mRNA in vitro. Then, the ribozyme was cloned into a vector under the control of U6 snRNA promoter, an RNA polymerase III promoter, for high rate of transcription in vivo. The vector was transfected into an interferon-beta producing recombinant CHO cell line, and some clones were screened out that exhibited low caspase-3 production by Western blot analysis. One such clone was then further analyzed and it showed good anti-apoptosis property with respect to cell viability, cell density, and interferon-beta production.

Animals↗